DNA Damage And Repair In Breast Cancer

乳腺癌每年占女性死亡总数的15%-18%，每年约有18万新确诊病例。尽管乳腺癌的原因尚不清楚，但有几条证据表明，DNA损伤的积累和DNA修复的缺陷在乳腺癌中发挥了重要作用。据推测，DNA碱基损伤可能会导致突变，从而可能致癌。最重要的是由活性氧(ROS)引起的碱基损伤。细胞DNA通过内源性细胞代谢或外源性环境诱变剂暴露于ROS。ROS可引起广泛的DNA损伤。胸腺嘧啶二醇(TG)和8-羟基鸟嘌呤(8-oxoG)是一些最有害的氧化碱基损伤。胸腺嘧啶二醇是一种有毒的病变，可以阻止DNA复制和转录，导致细胞死亡。8-oxoG是一种前突变损伤。为了避免8-oxoG的有害影响，生物体已经开发出修复这种损伤的机制。应用高效液相色谱和气相色谱-质谱仪的研究表明，浸润性导管癌组织中8-oxoG的水平高于正常乳腺组织，提示氧化损伤与乳腺癌的病因有关。研究表明，8-oxoG是通过碱基切除修复(BER)途径修复的。到目前为止，还没有关于去除乳腺癌细胞中8-oxoG或其他氧化性DNA碱基损伤的报道。因此，在乳腺癌发生过程中，氧化损伤的误码率是否发生改变仍有待确定。因此，我们假设，从正常到恶性的乳腺组织的转变可能是由于氧化DNA损伤修复的缺陷，从而导致重要基因的突变。这种缺陷可能发生在核和/或线粒体基因组中。线粒体DNA(MtDNA)编码13种参与氧化磷酸化的蛋白质。线粒体DNA氧化诱导的突变可导致线粒体功能障碍，并与退行性疾病、癌症和衰老有关。因此，有效的氧化损伤修复过程对于细胞维持线粒体基因组的完整性至关重要。我们检测了从非肿瘤性乳腺上皮细胞系和乳腺癌MCF-7和MDA-MB-468细胞系中提取的核和线粒体提取物从双链寡核苷酸中切割8-oxoG和TG损伤的能力。我们在这项研究中报道了三个重要的发现：第一，MCF-7和MDA-MB-468乳腺癌细胞株的线粒体提取物在清除8-oxoG方面都缺乏。与野生型相比，两种乳腺癌细胞株切割8-oxoG的能力都降低了两倍以上。这种缺陷是8-oxoG所特有的，因为用相同的线粒体提取液切割TG与野生型细胞相似。其次，两种乳腺癌细胞系的核提取液比线粒体提取液更快、更有效地去除8-oxoG。第三，核提取物比8-oxoG更快地清除甘油三酯。我们首次发现人类乳腺癌细胞系的线粒体在修复8-oxoG的过程中存在缺陷。这种8-oxoG的缺陷修复可能意味着乳腺癌细胞有很高的mtDNA突变发生率。这些乳腺癌细胞的线粒体DNA的遗传状态仍有待通过序列分析来确定。因此，我们得出结论，线粒体基因组中8-oxoG的修复可能在乳腺癌的发生发展中起关键作用。我们的研究可能为乳腺癌的新型分子干预提供基础。我们进一步提出，其他形式的癌症在氧化DNA损伤修复方面可能存在缺陷。我们还假设，这些细胞的线粒体DNA可能有过度的氧化损伤，导致氧化修复缺陷。为了解决这一假设，这些和其他乳腺癌细胞株的线粒体和基因组DNA将通过LC/GC质量光谱分析来确定基础水平的氧化损伤。我们还将评估通过用特定的氧化损伤剂(例如甲苯二酮、伽马射线或过氧化氢)处理细胞来诱导DNA氧化损伤，以便通过LC/GC质谱分析来分析损伤形成的速率。在我们最近的工作中，我们已经开始评估BRCA1基因在氧化损伤修复中的作用。我们正在使用两个BRCA-1突变纯合或杂合的细胞系(CRL2336和CRL2337)。该项目的wt对照是AG10009淋巴母细胞系。初步数据表明，与野生型细胞相比，BRCA-1突变纯合子细胞中氧化损伤、8-oxoG、胸腺嘧啶乙醇和5-羟基胞嘧啶的核修复减少。该突变细胞系中氧化损伤的线粒体修复与野生型细胞相当。 一旦我们确定了BRCA1突变细胞系的修复表型，将进一步研究参与氧化损伤修复的特定修复酶(例如胸腺嘧啶乙二醇人内切酶III(HNTH1))与BRCA1和BASC复合体(BRCA1相关基因组监视复合体)的其他成员是否如Wang et所定义的那样。等(BRCA1、ATM、NBS1、BLM、MRE-11、RAD50、MSH2、MLH1、MSH6)。BRCA1基因可能在乳腺组织的DNA氧化修复中发挥重要作用，可能部分解释了BRCA1基因在乳腺肿瘤发生中的作用。

Breast cancer accounts for 15-18% of all deaths among women every year, with about 180,000 new cases being diagnosed every year. Even though the causes of breast cancer remain unknown, several lines of evidence suggest that accumulation of DNA damage coupled with defects in DNA repair play an important role in breast cancer. It has been speculated that DNA base damage may lead to mutations that subsequently can be carcinogenic. Of primary importance are the base lesions caused by reactive oxygen species (ROS). Cellular DNA is exposed to ROS either endogenously by cellular metabolism or through exogenous exposure to environmental mutagens. ROS induce a wide range of DNA lesions. Thymine glycol (Tg) and 8-hydroxyguanine (8-oxoG) are some of the most deleterious oxidative base lesions. Thymine glycol is a toxic lesion that blocks DNA replication and transcription, causing cell death. 8-oxoG is a premutagenic lesion. In order to avoid the harmful effects of 8-oxoG, organisms have developed mechanisms for repairing this damage. Studies using High Performance Liquid Chromatography and Gas Chromatography-Mass Spectrometry have revealed increased levels of 8-oxoG in invasive ductal breast carcinomas relative to normal breast tissue implicating oxidative damages in the etiology of breast cancer. It has been shown that 8-oxoG is repaired via the base excision repair (BER) pathway. To date, there are no reports on the removal of 8-oxoG or other oxidative DNA base lesions in breast cancer cells. Therefore, it remains to be established whether BER of oxidative lesions is altered during breast carcinogenesis. We therefore, hypothesized that the transformation from normal to malignant breast tissue may result from defects in oxidative DNA damage repair, consequently leading to mutations in important genes. Such a defect may occur in the nuclear and/or the mitochondrial genome. Mitochondrial DNA (mtDNA) encodes 13 proteins that are involved in oxidative phosphorylation. Oxidatively induced mutations in the mtDNA can lead to dysfunctional mitochondria, and have been implicated in degenerative diseases, cancer and aging. Therefore, effective oxidative damage repair processes are essential in order for the cell to maintain the integrity of the mitochondrial genome. We examined the ability of nuclear and mitochondrial extracts from a non-neoplastic mammary epithelial cell line and breast cancer MCF-7 and MDA-MB-468 cell lines to incise 8-oxoG and Tg lesions from duplex oligonucleotides. We have reported three important findings in this study: first, mitochondrial extracts from both MCF-7 and MDA-MB-468 breast cancer cell lines are deficient in the removal of 8-oxoG. Both breast cancer cell lines exhibited more than two-fold decrease in their ability to incise 8-oxoG relative to the wild type. This defect was specific for 8-oxoG since the incision of Tg by the same mitochondrial extracts was comparable to that of wild type cells. Second, nuclear extracts from both breast cancer cell lines removed 8-oxoG more rapidly and efficiently than mitochondrial extracts. Third, nuclear extracts were shown to remove Tg more rapidly than 8-oxoG. We have shown for the first time that mitochondria from human breast cancer cell lines are defective in the repair of 8-oxoG. This defective repair of 8-oxoG may imply that breast cancer cells have a high incidence of mtDNA mutations. The genetic status of mtDNA from these breast cancer cells remains to be determined through sequence analyses. Therefore, we conclude that repair of 8-oxoG in the mitochondrial genome may be crucial in the development of breast cancer. Our studies may provide a basis for novel molecular interventions of breast cancer. We further propose that other forms of cancer may be defective in oxidative DNA damage repair. We have also hypothesized that mitochondrial DNA of these cells may have excessive oxidative damage caused by defective oxidative repair. To address this hypothesis, mitochondrial and genomic DNA from these and other breast cancer cell lines will be analyzed by LC/GC mass spectrophotometry to determine the basal level oxidative damage. We will also assess induction of oxidative DNA damage by treating cells with specific oxidative damaging agents ( e.g. Menadione, gamma irradiation, or hydrogen peroxide), for analysis of rates of lesion formation via LC/GC mass spectrophotometry. In our most recent work, we have begun to evaluate the role of the BRCA 1 gene in oxidative damage repair. We are using two cell lines (CRL2336 and CRL2337) that are either homozygous or heterozygous for BRCA-1 mutation. The wt control for this project is the AG10009 lymphoblast cell line. Preliminary data suggests that nuclear repair of oxidative lesions, 8-oxoG, thymine glycol and 5-hydroxycytosine is reduced in cells homozygous for the BRCA-1 mutation relative to wild-type cells. Mitochondrial repair of oxidative lesions in this mutant cell line is comparable to that of wild-type cells. Once we have confirmed the repair phenotype of the BRCA1 mutant cell lines, further investigation will be directed to examining whether the specific repair enzymes involved in oxidative lesion repair (e.g. human endonuclease III (hNTH1) for thymine glycol) complexes with BRCA1 and other members of the BASC complex (Brca1-associated genome surveillance complex) as defined Wang et. al.( BRCA1, ATM, NBS1, BLM, MRE-11, RAD50, MSH2, MLH1, MSH6 ). It is possible that the BRCA1 gene may play an important role in oxidative DNA repair in mammary tissue possibly partially explaining one its roles breast tumorigenesis.